Bioremediation of 2,4,6-Trinitrotoluene by Bacterial Nitroreductase

Improper handling, production, storage, and decommissioning of the polynitroaromatic explosive 2,4,6-trinitrotoluene (TNT) has led to extensive contam...
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Environ. Sci. Technol. 2008, 42, 7405–7410

Bioremediation of 2,4,6-Trinitrotoluene by Bacterial Nitroreductase Expressing Transgenic Aspen PIETER VAN DILLEWIJN,† ´ L. COUSELO,‡ JOSE ELENA CORREDOIRA,‡ ANTONIO DELGADO,§ ROLF-MICHAEL WITTICH,† ANTONIO BALLESTER,‡ AND J U A N L . R A M O S * ,† Estacioín Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, Department of Environmental Protection, Granada, Spain, Instituto de Investigaciones Agrobioloígicas de Galicia, Consejo Superior de Investigaciones Científicas, Santiago de Compostela, Spain, Estacioín Experimental del Zaidiín, Consejo Superior de Investigaciones Científicas, Department of Environmental Geochemistry, Granada, Spain

Received May 16, 2008. Revised manuscript received July 15, 2008. Accepted July 25, 2008.

Trees belonging to the genus Populus are often used for phytoremediation due to their deep root formation, fast growth and high transpiration rates. Here, we study the capacity of transgenic hybrid aspen (Populus tremula x tremuloides var. Etropole) which expresses the bacterial nitroreductase gene, pnrA, to tolerate and take-up greater amounts of the toxic and recalcitrant explosive, 2,4,6-trinitrotoluene (TNT) from contaminated waters and soil. Transgenic aspen tolerate up to 57 mg TNT/L in hydroponic media and more than 1000 mg TNT/ kg soil, whereas the parental aspen could not endure in hydroponic culture with more than 11 mg TNT/L or soil with more than 500 mg TNT/kg. Likewise, the phytotoxicological limit for transgenic plants to a constant concentration of TNT was 20 mg TNT/L while wild-type plants only tolerated 10 mg TNT/L. Transgenic plants also showed improved uptake of TNT over wild-type plants when the original TNT concentration was above 35 mg TNT/L in liquid media or 750 mg TNT/kg in soil. Assays with 13Clabeled TNT show rapid adsorption of TNT to the root surface followed by a slower entrance rate into the plant. Most of the 13C-carbon from the labeled TNT taken up by the plant (>95%) remains in the root with little translocation to the stem. Altogether, transgenic aspen expressing pnrA are highly interesting for phytoremediation applications on contaminated soil and underground aquifers.

Introduction Improper handling, production, storage, and decommissioning of the polynitroaromatic explosive 2,4,6-trinitrotoluene (TNT) has led to extensive contamination of soil and * Corresponding author phone: +34 958 181608; fax: +34 958 135740; e-mail: [email protected]. † Department of Environmental Protection. ‡ Consejo Superior de Investigaciones Científicas. § Department of Environmental Geochemistry. 10.1021/es801231w CCC: $40.75

Published on Web 08/27/2008

 2008 American Chemical Society

groundwater (1, 2). This xenobiotic is toxic to humans, animals, plants, and microorganisms and is recalcitrant to degradation (1, 2). To clean up contaminated sites, phytoremediation with plants is receiving increased interest (3, 4). For effective phytoremediation, one should make use of plant species that tolerate contaminants and have the ability to remove large amounts of target chemicals. The use of tree species for phytoremediation (dendroremediation) has several advantages over smaller plants such as large biomass, long life cycle, low nutrient requirements, and intrinsic resistance to many pollutants (5). Trees belonging to the genus Populus are especially useful for phytoremediation because of their deep extensive root system, high water uptake, and rapid growth (6). This genus has also successfully been engineered genetically for phytoremediation purposes (7, 8). Moreover, poplar trees have been shown to resist up to 5 mg TNT/L and remove this xenobiotic from the medium (9-12). In general, plants appear to deal with TNT as a “green liver”, whereby the contaminant is detoxified and sequestered within plant tissues rather than mineralized to carbon dioxide and nitrogen (13, 14). Detoxification occurs by transforming the chemical, conjugating it to plant metabolites, and then sequestering the resulting macromolecules into vacuoles or polymers such as lignin (15-17). As a result, in most terrestrial plants TNT and its derivatives accumulate in the roots, and to a lesser extent, are transported to the stem and leaves. Similarly, in poplar plants, experiments conducted with 14CTNT showed that about 75% of the radiolabeled carbon remained in roots and lower stems, whereas only about 10% was translocated to the upper stem and leaves (9). TNT transformation by plants consists mainly of the sequential reduction of the nitro side groups of the molecule by the plant’s endogenous nitroreductases to hydroxylamine intermediates and then to amino derivatives. In poplar plants, aminodinitrotoluenes and as yet unidentified polar metabolites, which are probably the result of reduced TNT derivatives conjugated to plant metabolites, have been detected (9). The conversion of TNT to hydroxylamine derivatives is rate limiting in Arabidopsis thaliana, which suggests that enzymes catalyzing this reaction are good candidates for additional study to improve TNT removal by plants (18). Microorganisms have an arsenal of nitroreductases, which efficiently reduce the nitro side groups of TNT to different isomers of aminonitrotoluenes, which is interesting because these products can bind irreversibly to clay and organic material in the soil (19). These enzymes also attack other nitroaromatic compounds, which could be present in contaminated sites. Therefore, in order to improve the phytoremediation capability of plants, bacterial nitroreductases could be engineered into plant genomes. This has been performed with success by Hannink et al. (20) who obtained transgenic tobacco expressing the nitroreductase gene, nfsI, from Enterobacter cloacae. Similarly, Kurumata et al. (21) engineered Arabidopsis thaliana plants to express the nitroreductase, nfsA, of Escherichia coli. These transgenic plants resisted higher concentrations of TNT than did its wild-type counterparts. Here, we improve upon this theme by using a tree species as the target plant. We describe transgenic aspen (Populus tremula × tremuloides var. Etropole) which express the gene encoding the nitroreductase PnrA of Pseudomonas putida (22). This nitroreductase reduces TNT to 4-hydroxylamino-2,6-dinitrotoluene at a very high rate, and also reduces a broad range of other nitroaromatic compounds (22). Here, we study the capacity of the transgenic aspen to tolerate and take-up TNT from hydroponic media VOL. 42, NO. 19, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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and soil and provide experimental evidence for improved phytoremediation by these transgenic trees.

Experimental Section Chemicals. TNT (>99% pure) was obtained from Unio´n Espan ˜ ola de Explosivos (Madrid, Spain). Standards were obtained from AccuStandard (New Haven, CT). 13C-TNT was synthesized from >98% pure [ring-U-13C] toluene as described in ref 23. Bacterial Strains. Pseudomonas putida JLR11, and Agrobacterium tumefaciens C58C1 (pBIpnrA) strains were grown at 28 °C in LB medium (24). Plant Material, Transformation, and Molecular Analyses. Stock shoot cultures of aspen subcultured at 6 week intervals on MS medium (25) were used as a source for explants. Shoot multiplication of both wild type and transgenic explants was as described in ref 26. All cultures were kept in a growth chamber with 16 h of light provided by cool-white fluorescent lamps (flux density of 50-60 µmol · m-2 · s-1) and a temperature of 25 °C. In vitro rooted plants were subjected to acclimatization in a tunnel in a greenhouse and after approximately 9-10 weeks were used for hydroponic and soil experiments. Internodal segments of in vitro micropropagated aspen shoots were transformed by coculture with A. tumefaciens C58C1 (strain containing binary vector pBIpnrA (Supporting Information)) as described in ref 26. To confirm the presence of the pnrA gene, PCR with the primer pair 5′-AGCCAGCTAACTTACCTGC-3′ and 5′-CTCATCCTTCGGTCATAGG-3′ and Southern blot hybridization with pnrA were done using DNA isolated from leaves of both transgenic and untransformed plants. Each successfully transformation event constituted a transgenic line. Quantative two-step realtime RT-PCR was used to determine expression of the transgene in different tissues (see Supporting Information). Hydroponic Assays. To determine the concentration of TNT tolerated by wild type and transgenic plants, opaque covered containers containing 2.5 L of Hoagland medium (27) and an aeration system were used. To determine the uptake and toxicity of TNT to wild type and transgenic plants, the effect on transpiration was measured in a gravitational hydroponic system consisting of 250 mL graduated cylinders covered with aluminum foil (to protect roots against light) and filled with Hoagland medium. The roots (10-15 cm) of acclimatized plants were rinsed gently and introduced into either containers or cylinders before fastening with foam rubber plugs. The plants were acclimatized in the growth chamber for 2 days prior to each experiment. Growth chamber conditions were 60% humidity, 14:10 h light: dark photoperiod, 24:18 °C day:night temperature. Each experiment was initiated by replacing the Hoagland medium with medium spiked with different concentrations of TNT. The tolerance assay consisted of exposing four plants to Hoagland medium spiked with a range of TNT concentrations from 0 to 57 mg TNT/L and measuring plant length at a regular basis for 28 days and finally dry weights when the experiment finished. For uptake experiments, three to four plants were exposed to Hoagland medium spiked with 20, 35, or 50 mg TNT/L, and TNT was measured for 48 h. The TNT half-life was calculated using the slope of the curve multiplied by half the value of the original TNT concentration. For the toxicity experiments, the transpiration of four plants was monitored volumetrically for 2 weeks. These plants were exposed to a relatively constant concentration of TNT by replacing the Hoagland medium spiked with 0, 5, 10, 15, 20, 35, or 50 mg TNT/L every two days. In this experiment, the relative transpiration, RT, was calculated according to RT ) Tr/Tr0 by which the amount of medium transpired by each plant (in mL) at each time point (Tr) was compared to the 7406

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mean amount (in mL) transpired by plants of the same line but in control uncontaminated medium (Tr0) at the same time point. To determine TNT and its transformation products in plant organs, at least six plants were exposed for 48 h to Hoagland medium saturated with TNT (approximately 113.5 mg TNT/L). Then, the roots were separated from the shoots and washed three times for 5 min with 20% (v/v) methanol/ water to remove TNT or its metabolites which had adsorbed to the root surface. Prior to freezing at -80 °C each plant part was weighed. The extraction procedure and hydrolyses of extracts is described in the Supporting Information. Soil Assays. To determine the tolerance of wild type and transgenic plants in soil, the roots of three acclimatized 10-15 cm long plants were rinsed gently and placed in plant pots filled with 1800-2000 g soil spiked with different quantities of powdered TNT. The soil used has the following characteristics: 38% sand; 43% silt; and 19% clay, pH 7.9. The organic matter content was 2.1%, and its CaCO3 content was 8%. Plants were watered regularly with tap water and fertilized once-weekly with Hoagland medium. To mitigate possible leaching of TNT or its metabolites in the soil column, watering was limited to the water-holding capacity of the soil (approximately 30%). Plant length was measured regularly, and at the start and end of the experiment the TNT concentration in the soil was determined. Analytical Methods. To determine the concentration of TNT and its transformed derivatives in liquid media, 1 mL samples were passed through a GHP Acrodisc 0.45 µm syringe filter (VWR International, Barcelona, Spain) prior to analyses by high performance liquid chromatography (HPLC). To determine the concentration of TNT and its transformed products in bulk or rhizosphere soil a modified version of the U.S. Environmental Protection Agency (USEPA) 8330 method was used (see Supporting Information). For HPLC, a Hewlett-Packard 1050 chromatograph with a diode array detector and a reversed phase column (Novapak 5 µm, C8, 150 × 3.9 mm, Waters S.A.) was employed. TNT and its metabolites were measured as described in ref 28. 13C-TNT Experiments. To determine the incorporation of 13C-TNT into plant material, plants (three replicates) were placed into 100 mL flasks filled with 60 mL of Hoagland solution containing 35 mg 13C-TNT/L. TNT concentration was measured at regular intervals. Similarly, whole plants were removed at regular intervals for analysis. Root material was washed three times for 5 min with 20% methanol/water (v/v) to remove adsorbed TNT or derived metabolites. All plant material was dried at 55 °C for 72 h prior to analysis in an elemental analyzer-isotope ratio mass spectrometer online with a Delta Plus XL mass spectrometer. The overall precision of analyses was (0.1‰ for 13C. For the calculation of the stable isotope composition, the absorption index and the calculation of 13C-TNT in roots, see the Supporting Information. Statistical Analysis. For statistical analysis, the Excel 2003 and program GraphPad InStat 3.06 programs were used. To measure statistical significance between two data sets, onetailed unpaired t tests with Welch correction were applied with significant difference given when p < 0.05. All error calculations represent standard error at 95% confidence.

Results and Discussion Construction of Transgenic Aspen. Thirty independent pnrA transgenic aspen lines were obtained by Agrobacteriummediated transformation. The line with the highest mRNA levels of pnrA (relative to 18S RNA) was selected for use in subsequent experiments. Characterization of the transcription pattern of pnrA in each organ of this transgenic line showed more relative cDNA quantities in leaves (2.58 ( 0.25)

FIGURE 1. Comparison of aspen grown in hydroponic media with different TNT concentrations for 28 days. Photographs of (A) wild-type aspen and (B) pnrA expressing aspen. () Shoot dry weights and lengths of (D) wild type and transgenic (E) plants in hydroponic medium growing in hydroponic medium with different TNT concentrations after 28 days. All standard error bars at p < 0.05, n ) 4. and stems (2.15 ( 0.27) than in roots (1.0 ( 0.24). Single copy insertion of the transgene was confirmed by Southern-blot analysis. Tolerance of Transgenic Aspen to TNT. Experiments under hydroponic and soil conditions were performed to compare the tolerance of wild-type and transgenic trees to different concentrations of TNT. In hydroponic assays, the growth and health of wild-type plants were drastically affected at concentrations above 11 mg TNT/L (Figure 1A, C and D). Although pnrA expressing plants were also affected at this concentration, these plants continued to show growth in 57 mg TNT/L (Figure 1B, C, and E). In contaminated soil, the growth of all plants was affected at 250 mg TNT/ kg soil (Figure 2), and in the case of wild-type plants, completely inhibited at concentrations greater than 500 mg TNT/ kg soil (Figure 2A, C, and D). However, pnrA transgenic aspen could withstand and still grow at 1000 mg TNT/ kg soil (Figure 2B, C, and E). As a result, after 56 days, the dry weight of pnrA transgenic aspen tended to be higher at concentrations of 500 mg TNT/kg and above, and significantly higher (p < 0.05) at 1000 mg TNT/ kg soil, compared to wild-type plants (Figure 2C). Altogether, the results show that pnrA expression increases aspen tolerance toward higher TNT concentrations in both hydroponic media and soil. Thompson et al. (11) determined that larger, more mature poplar plants could resist higher concentrations of TNT. To test this observation, hydroponic assays were performed using aspen with approximately twice the age of the plants described in the assays above. After 28 days, both wild type and transgenic plants could grow (1.53 ( 1.01 and 3.5 ( 1.07 cm, respectively (n g 4)) in 68 mg TNT/L, a growth inhibitory concentration for younger plants. pnrA expressing aspen continued to show growth (4.17 ( 0.61 cm) even at 91 mg TNT/L, whereas wild-type plants, though still alive, did not

(0.3 ( 0.11 cm). This has important implications for in situ phytoremediation as it implies that the use of more mature transgenic plants will allow the remediation of soils contaminated with higher concentrations of TNT than reported here. TNT Uptake by Transgenic Aspen. The amount and rate of TNT uptake was measured as well for transgenic and wildtype aspen. In the hydroponic experiment to determine the resistance of each type of plant to TNT (Figure 1), TNT was undetectable within 4 days in all concentrations (data not shown). Depending on the initial TNT concentration, the amount of aminodinitrotoluenes (ADNTs) peaked at 7 days with 1.6-4.4 mg ADNT/L (4ADNT and 2ADNT) to slowly decrease to between 0 and 2.8 mg ADNT/L after 28 days. Other TNT metabolites such as hydroxylaminodinitrotoluenes (HADNTs), azoxynitrotoluenes or recently described diarylhydroxylamines or diarylamines (28) were not detected in the medium. To study TNT removal in detail, initial TNT concentrations of 20, 35, or 50 mg TNT/L were used (Figure 3A). In the absence of plants, the concentration of TNT changed little throughout the experiment. When exposed to 20 or 35 mg TNT/L, TNT was taken up just as rapidly by wild-type plants as by transgenic plants (TNT half-life of approximately 30 and 20 h, for 20 and 35 mg TNT/L, respectively). However, when exposed to 50 mg TNT/L, pnrA transgenic aspen took up TNT more quickly (TNT half-life of approximately 30 h) than wild-type plants (TNT half-life of approximately 50 h). In these experiments only negligible amounts of TNT transformation products could be detected. The slower TNT uptake by wild-type plants could be due to TNT toxicity. If so, this suggests that the expression of pnrA helps the plant to maintain high TNT uptake under otherwise restrictive conditions. VOL. 42, NO. 19, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 2. Growth of aspen in soil contaminated with different concentrations of TNT after 56 days. Photographs of (A) wild-type and (B) pnrA expressing aspen. (C) Shoot dry weight and lengths of (D) wild type and (E) pnrA expressing aspen in soil after 56 days. For all graphs standard error bars are given at a confidence interval of p < 0.05 of three repetitions (n ) 3). TNT removal was also determined in rhizosphere soil after 56 days. The percentage of TNT removal in soil by wild-type plants decreased as the level of the contaminant increased (Figure 3B). In contrast, transgenic pnrA expressing aspen removed over 60% of the original amount of TNT in soil. As a result, TNT removal by pnrA expressing plants became more important as the concentration of TNT in the soil increased (i.e., g750 mg TNT/ kg soil). It should be mentioned that in unplanted soils up to 50% TNT can become adsorbed to humic acids and organic and inorganic soil material after 56 days (19) with the consequent reduction in the bioavailability of TNT for plants. This observation is in agreement with Thompson et al. (9, 11) and constitutes a bottleneck for effective phytoremediation; nonetheless, this limitation can be most probably alleviated using long-lived trees to ensure a more continuous removal of unbound TNT as it is leached from soil by irrigation. With respect to the presence of TNT transformation products in soil, only ADNTs comprising of 2ADNT (about 33% w/w of the total amount of ADNTs detected), and 4ADNT (about 66%) were detected in either rhizosphere or bulk soil. The quantity of ADNTs detected in unplanted bulk soil (up to 1.6 mg ADNT/kg soil) was below that measured in the rhizosphere of aspen. The level of ADNTs tended to be higher in the rhizosphere of wild-type plants (up to 4.8 mg ADNT/kg soil) than of pnrA expressing plants (up to 3.6 mg ADNT/ kg soil). If these differences are due to plant uptake or the action of rhizospheric microbial communities remains unclear. More importantly, the results from both hydroponic and soil experiments demonstrate the greater capacity of transgenic aspen to remove TNT from highly contaminated media. This is in agreement with results obtained with bacterial nitroreductase expressing transgenic 7408

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tobacco, which also removed significantly larger quantities of TNT from liquid media (20) and soil (29) than wild-type plants. Phytotoxicological Limits of Transgenic Aspen. To determine differences in the phytotoxicological limits between wild-type aspen and transgenic plants, transpiration was monitored in hydroponic medium in which the concentration of TNT was kept constant. Transpiration rates were used as a parameter because this is more sensitive for determining toxicity in plants than measuring growth or increases in dry weight (11). Our results show that wild-type aspen failed to tolerate a constant concentration of TNT above 10 mg TNT/L, whereas pnrA expressing aspen were only affected at constant concentrations above 20 mg TNT/L (Supporting Information Figure S1). Thompson et al. (11) showed that TNT exerted phytotoxicological effects on poplar cuttings at 5 mg TNT/L. The wild-type aspen used in this work appear to withstand double this amount of TNT and pnrA expression in these trees improves resistance by another 2-fold. Fate of TNT in Plant Tissues. An important concern for phytoremediation of TNT is the fate of the contaminant once it is taken up by the plant. Extraction of roots or shoots of plants used for the above-mentioned experiments did not reveal any clear identification of TNT, transformation products, or products upon hydrolysis of potential conjugates. This indicates that TNT is quickly transformed and sequestered by aspen. Only when plants were exposed for 2 days to liquid medium saturated with TNT was TNT, 4HADNT, 4ADNT, and traces of 2ADNT detected in roots (Figure 4, unhydrolyzed samples) but none in shoots. Azoxynitrotoluenes, diarylhydroxylamines, or diarylamines were not de-

FIGURE 3. Removal of TNT (uptake) by wild-type and pnrA expressing aspen from liquid medium and soil. (A) Uptake of TNT by wild-type and pnrA transgenic aspen exposed to hydroponic medium containing 20 mg TNT/L, 35 mg TNT/L and 50 mg TNT/L (n ) 4). Dotted lines indicate controls without plants. (B) Percentage of original TNT (at T ) 0) removed in unplanted soil or soil with wild type or pnrA expressing aspen (n ) 3). For all graphs standard error bars are given at a confidence interval of p < 0.05.

FIGURE 4. TNT and its transformation products measured in unhydrolyzed or hydrolyzed, solvent extracted root extracts of wild-type plants (wt) or transgenic plants (pnrA) which had been exposed to TNT for 48 h (standard error bars p < 0.05, n g 6). tected in any tissue. The concentration of HADNT was higher and of TNT lower in the roots of transgenic plants than in wild-type plants. This could be indicative of increased reduction due to pnrA expression, but these differences were not statistically significant (p < 0.05, Figure 4, unhydrolyzed samples). Although conjugated TNT derivatives have been described in Arabidopsis thaliana, Catharanthus roseus, and tobacco (15, 17, 18), the only indication for their presence in Populus has been as unknown polar transformation products (9). In our study, polar peaks, which disappeared upon acid hydrolysis (which could indicate putative conjugates), were not detected in the HPLC chromatograms of either wild type or transgenic root extracts. Moreover, when

FIGURE 5. (A) Distribution pattern of 13C enrichment from labeled TNT in roots and shoots of transgenic and wild type aspen. Plants were incubated with 13C-TNT and at the indicated times removed and treated as described in the Experimental Section. The δ13C values were determined from roots and shoots by mass spectrometry and compared using an absorption index to the natural δ13C levels present in tissues of control plants. (B) Adsorption and the subsequent incorporation of 13C from labeled TNT in the root of wild type and transgenic aspen. The percent (w/w) of the initial amount of 13C-TNT present in the flask was determined by measuring the amount of 13C-TNT in liquid media (closed symbols) by HPLC and the quantity of 13C from labeled TNT incorporated into roots (open symbols) as described in the Experimental Section at different time points. All error bars represent standard error (p < 0.05) for three repetitions (n ) 3). the amount of TNT together with the transformed products detected in either hydrolyzed or unhydrolyzed extracts are summed up, their total values are similar (Figure 4) indicating that little conjugate formation had occurred. As the plants do not tolerate liquid media saturated with TNT (transpiration stopped within 24 h), possibly the metabolic activity required for conjugate formation was compromised as well. This suggests that the conditions need to be optimized to study conjugate formation and characterization in aspen in greater detail; however, this is not within the scope of the present work. Comparison with the products detected in other transgenic plants expressing bacterial nitroreductases revealed different tendencies. In Arabidopsis, more ADNT but less TNT was observed in nfsA-expressing transgenic plants than in wild-type plants, but possible conjugates were not studied (21). In wild-type tobacco, TNT and ADNT appeared mostly in roots and only very little in shoots. However, no TNT and little ADNT were detected in the roots and shoots of nfsI transgenic tobacco plants (20). More recently, TNT transformation studies after short (10 h) exposures with seedlings of the same transgenic tobacco variant revealed the presence of 4HADNT and greater amounts of conjugates related to this metabolite than in wild-type roots indicating enhanced detoxification by the transgenic line (30). Distribution and Absorption of 13C-TNT. To obtain information of the distribution of TNT in tissues of either transgenic or wild-type plants, 13C ring labeled TNT was used. The experiment was performed with only 35 mg/L of labeled TNT to diminish the possible toxic effect on absorption by plants while maintaining concentrations high enough for detection of the stable isotope, 13C. As expected, 13C enrichment was observed principally in roots with only very little in the shoot compartment (2-6%) (Figure 5A). Moreover, VOL. 42, NO. 19, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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similar to TNT uptake results in liquid medium (Figure 3), no significant differences were observed in the absorption of 13C-TNT by wild type or transgenic aspen nor in the disappearance of TNT or its products (principally 4ADNT) in the medium (Figure 5B). Data in Figure 5B show that the speed of TNT disappearance from the medium is relatively fast, with nearly 100% of the initial TNT having disappeared from the hydroponic culture after 24 h. However, this contrasts with the rather slow absorption of 13C-TNT into the roots which accounts for approximately 30% of the initial amount of TNT after 4 days. Only 0.04% of the added TNT adhered to glassware and very little was transported to the shoot (Figure 5A) thereby diminishing the possibility carbon from the labeled TNT being evolved as CO2. We suggest that the remaining 13C-TNT was removed from root surfaces by washing with diluted methanol. In this way, the results indicate that adsorption of TNT or its derivatives to the root surface is a rapid process compared to the much slower incorporation (absorption) of the nitroaromatics into the plant tissue. As a result, probably more TNT would be taken up into the roots if plants had been left for a longer period of time. Taken together, the data presented in our work suggest that the expression of the bacterial nitroreductase pnrA, improved the natural capacity of aspen to tolerate, grow, and more importantly, eliminate TNT not only from contaminated hydroponic medium but also from contaminated soil where bioavailability is reduced. The advantageous characteristics of pnrA expressing aspen described in this work make these trees potentially excellent candidates for in situ TNT dendroremediation and serve as a proof of concept for the manipulation of other tree species better adapted to other climates and soil conditions.

Acknowledgments We thank M. Rey (Universidad de Vigo, Spain) for his expert advice and technical support on real-time PCR, and David Martı´n for excellent technical assistance. This study was supported by grants from the EU (MADOX QLRT- 200100345), MEC, Spain (AGL2005-00709) and Xunta de Galicia (PGIDIT03BTF40001PR). P.v.D. and J.L.C. were supported by I3P grants from CSIC.

Supporting Information Available Additional details concerning pBIpnrA binary plasmid construction, the quantification of pnrA expression in transgenic plants, the determination of TNT and its derivatives in soil and plant tissues, and calculations for data from experiments with 13C-TNT. Figure S1 shows phytotoxicological limits of transgenic and wild type plants to TNT. This material is available free of charge via the Internet at http://pubs.acs.org.

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